Patent Grant
7056701
The present invention relates to new biologically active polypeptides, their preparation and pharmaceutical compositions containing them.
More particularly, the present invention relates to essentially recombinant polypeptides composed of an active part derived from a natural or artificial polypeptide having a therapeutic activity and coupled to an albumin or to a variant of albumin. It is understood that the therapeutic activity of the polypeptides of the invention can be either direct (treatment of diseases), or indirect (and for example capable of being used in the prevention of diseases, in the design of vaccines, in medical imaging techniques and the like).
It is understood in the following text that the albumin variants designate any protein with a high plasma half-life which is obtained by modification (mutation, deletion and/or addition), by genetic engineering techniques, of a gene encoding a given isomorph of human serum albumin, as well as any macromolecule with a high plasma half-life obtained by in vitro modification of the protein encoded by such genes. Albumin being highly polymorphic, numerous natural variants have been identified and classified [Weitkamp L. R. et al., Ann. Hum. Genet. 37 (1973) 219].
The aim of the present invention is to prepare artificial proteins which are biologically active and can be used pharmaceutically. Indeed, numerous polypeptides possessing one or more potential therapeutic activities cannot be exploited pharmaceutically. This may have various reasons, such as especially their low stability in vivo, their complex or fragile structure, the difficulty of producing them on an industrially acceptable scale and the like. Likewise, some polypeptides do not give the expected results in vivo because of problems of administration, of packaging, of pharmacokinetics and the like.
The present invention makes it possible to overcome these disadvantages. The present invention indeed provides new molecules which permit an optimal therapeutic exploitation of the biological properties of these polypeptides. The present invention results especially from the demonstration that it is possible to couple genetically any active structure derived from a biologically active polypeptide to another protein structure consisting of albumin, without impairing the said biological properties thereof. It also results from the demonstration by the Applicant that human serum albumin makes it possible efficiently to present the active structure to its sites for interaction, and that it provides a high plasma stability for the polypeptide of the invention. The polypeptides of the invention thus make it possible to maintain, in the body, a given biological activity for a prolonged period. They thus make it possible to reduce the administered doses and, in some cases, to potentiate the therapeutic effect, for example by reducing the side effects following a higher administration. The polypeptides of the invention make it possible, in addition, to generate and to use structures derived from biologically active polypeptides which are very small and therefore very specific for a desired effect. It is understood that the peptides having a biological activity, which are of therapeutic interest, may also correspond to non-natural peptide sequences isolated for example from random peptide libraries. The polypeptides of the invention possess, moreover, a particularly advantageous distribution in the body, which modifies their pharmacokinetic properties and favours the development of their biological activity and their use. In addition, they also have the advantage of being weakly or non-immunogenic for the organism in which they are used. Finally, the polypeptides of the invention can be expressed (and preferentially secreted) by recombinant organisms, at levels permitting their industrial exploitation.
One subject of the present invention therefore relates to polypeptides containing an active part derived from a polypeptide having a therapeutic activity, coupled to an albumin or a variant of albumin.
In a specific embodiment, the peptides possessing a therapeutic activity are not of human origin. For example, there may be mentioned peptides, or their derivatives, possessing properties which are potentially useful in the pathologies of the blood and interstitial compartments, such as hirudin, trigramine, antistatine, tick anticoagulant peptides (TAP), arietin, applagin and the like.
More particularly, in the molecules of the invention, the polypeptide having a therapeutic activity is a polypeptide of human origin or a molecular variant. For example, this may be all or part of an enzyme, an enzyme inhibitor, an antigen, an antibody, a hormone, a factor involved in the control of coagulation, an interferon, a cytokine [the interleukins, but also their variants which are natural antagonists of their binding to the receptor(s), the SIS (small induced secreted) type cytokines and for example the macrophage inflammatory proteins (MIPs), and the like], of a growth factor and/or of differentiation [and for example the transformant growth factors (TGFs), the blood cell differentiation factors (erythropoietin, M-CSF, G-CSF, GM-CSF and the like), insulin and the growth factors resembling it (IGFs), or alternatively cell permeability factors (VPF/VEGF), and the like], of a factor involved in the genesis/resorption of bone tissues (OIF and osteospontin for example), of a factor involved in cellular motility or migration [and for example autocrine motility factor (AMF), migration stimulating factor (MSF), or alternatively the scatter factor (scatter factor/hepatocyte growth factor)], of a bactericidal or antifungal factor, of a chemotactic factor [and for example platelet factor 4 (PF4), or alternatively the monocyte chemoattracting peptides (MCP/MCAF) or neutrophil chemoattracting peptides (NCAF), and the like], of a cytostatic factor (and for example the proteins which bind to galactosides), of a plasma (and for example von Willebrand factor, fibrinogen and the like) or interstitial (laminin, tenascin, vitronectin and the like) adhesive molecule or extracellular matrices, or alternatively any peptide sequence which is an antagonist or agonist of molecular and/or intercellular interactions involved in the pathologies of the circulatory and interstitial compartments and for example the formation of arterial and venous thrombi, cancerous metastases, tumour angiogenesis, inflammatory shock, autoimmune diseases, bone and osteoarticular pathologies and the like.
The active part of the polypeptides of the invention may consist for example of the polypeptide having a whole therapeutic activity, or of a structure derived therefrom, or alternatively of a non-natural polypeptide isolated from a peptide library. For the purposes of the present invention, a derived structure is understood to mean any polypeptide obtained by modification and preserving a therapeutic activity. Modification should be understood to mean any mutation, substitution, deletion, addition or modification of genetic and/or chemical nature. Such derivatives may be generated for various reasons, such as especially that of increasing the affinity of the molecule for its binding sites, that of improving its levels of production, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or alternatively of reducing its side effects, or that of conferring on it new biological properties. As an example, the chimeric polypeptides of the invention possess pharmacokinetic properties and a biological activity which can be used for the prevention or treatment of diseases.
Particularly advantageous polypeptides of the invention are those in which the active part has:
(a) the whole peptide structure or,
(b) a structure derived from (a) by structural modification (mutation, substitution addition and/or deletion of one or more residues) and possessing a therapeutic activity.
Among the structures of the (b) type, there may be mentioned more particularly the molecules in which certain N- or O-glycosylation sites have been modified or suppressed, the molecules in which one or more residues have been substituted, or the molecules in which all the cystein residues have been substituted. There may also be mentioned molecules obtained from (a) by deletion of regions not involved or not highly involved in the interaction with the binding sites considered, or expressing an undesirable activity, and molecules containing, compared to (a), additional residues such as for example an N-terminal methionine and/or a signal for secretion and/or a joining peptide.
The active part of the molecules of the invention can be coupled either directly or via an artificial peptide to albumin. Furthermore, it may constitute the N-terminal end as well as the C-terminal end of the molecule. Preferably, in the molecules of the invention, the active part constitutes the C-terminal part of the chimera. It is also understood that the biologically active part may be repetitive within the chimera. A schematic representation of the molecules of the invention is given in
Another subject of the invention relates to a process for preparing the chimeric molecules described above. More specifically, this process consists in causing a eukaryotic or prokaryotic cellular host to express a nucleotide sequence encoding the desired polypeptide, and then in harvesting the polypeptide produced.
Among the eukaryotic hosts which can be used within the framework of the present invention, there may be mentioned animal cells, yeasts or fungi. In particular, as regards yeasts, there may be mentioned yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces, or Hansenula. As regards animal cells, there may be mentioned COS, CHO and C127 cells and the like. Among the fungi capable of being used in the present invention, there may be mentioned more particularly Aspergillus ssp, or Trichoderma ssp. As prokaryotic hosts, the use of bacteria such as Escherichia coli, or belonging to the genera Corynebacterium, Bacillus, or Streptomyces is preferred.
The nucleotide sequences which can be used within the framework of the present invention can be prepared in various ways. Generally, they are obtained by assembling, in reading phase, the sequences encoding each of the functional parts of the polypeptide. The latter may be isolated by the techniques of persons skilled in the art, and for example directly from cellular messenger RNAs (mRNAs), or by recloning from a complementary DNA (cDNA) library, or alternatively they may be completely synthetic nucleotide sequences. It is understood, furthermore, that the nucleotide sequences may also be subsequently modified, for example by the techniques of genetic engineering, in order to obtain derivatives or variants of the said sequences.
More preferably, in the process of the invention, the nucleotide sequence is part of an expression cassette comprising a region for initiation of transcription (promoter region) permitting, in the host cells, the expression of the nucleotide sequence placed under its control and encoding the polypeptides of the invention. This region may come from promoter regions of genes which are highly expressed in the host cell used, the expression being constitutive or regulatable. As regards yeasts, it may be the promoter of the gene for phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GPD), lactase (LAC4), enolases (ENO), alcohol dehydrogenases (ADH), and the like. As regards bacteria, it may be the promoter of the right-hand or left-hand genes from the lambda bacteriophage (PL, PR), or alternatively the promoters of the genes for the tryptophan (Ptrp) or lactose (Plac) operons. In addition, this control region can be modified, for example by in vitro mutagenesis, by the introduction of additional control elements or of synthetic sequences, or by deletions or substitutions of the original control elements. The expression cassette may also comprise a region for termination of transcription which is functional in the host envisaged, positioned immediately downstream of the nucleotide sequence encoding a polypeptide of the invention.
In a preferred mode, the polypeptides of the invention result from the expression, in a eukaryotic or prokaryotic host, of a nucleotide sequence and from the secretion of the product of expression of the said sequence into the culture medium. It is indeed particularly advantageous to be able to obtain, by the recombinant route, molecules directly in the culture medium. In this case, the nucleotide sequence encoding a polypeptide of the invention is preceded by a “leader” sequence (or signal sequence) directing the nascent polypeptide in the secretory pathways of the host used. This “leader” sequence may be the natural signal sequence of the biologically active polypeptide in the case where the latter is a naturally secreted protein, or that of the stabilizing structure, but it may also be any other functional “leader” sequence, or an artificial “leader” sequence. The choice of one or the other of these sequences is especially guided by the host used. Examples of functional signal sequences include those of the genes for the sexual pheromones or the “killer” toxins of yeasts.
In addition to the expression cassette, one or several markers which make it possible to select the recombinant host may be added, such as for example the URA3 gene from the yeast S. cerevisiae, or genes conferring the resistance to antibiotics such as geneticin (G418) or to any other toxic compound such as certain metal ions.
The unit formed by the expression cassette and by the selectable marker can be introduced directly into the considered host cells, or previously inserted in a functional self-replicating vector. In the first case, sequences homologous to regions present in the genome of the host cells are preferably added to this unit; the said sequences then being positioned on each side of the expression cassette and of the selectable gene so as to increase the frequency of integration of the unit into the genome of the host by targetting the integration of the sequences by homologous recombination. In the case where the expression cassette is inserted in a replicative system, a preferred replication system for yeasts of the genus Kluyveromyces is derived from the plasmid pKDI originally isolated from K drosophilarum; a preferred replication system for yeasts of the genus Saccharomyces is derived from the 2μ plasmid from S. cerevisiae. Furthermore, this expression plasmid may contain all or part of the said replication systems, or may combine elements derived both from the plasmid pKD1 and the 2μ plasmid.
In addition, the expression plasmids may be shuttle vectors between a bacterial host such as Escherichia coli and the chosen host cell. In this case, a replication origin and a selectable marker functioning in the bacterial host are required. It is also possible to position restriction sites surrounding the bacterial and unique sequences on the expression vector: this makes it possible to suppress these sequences by cutting and religation in vitro of the truncated vector before transformation of the host cells, which may result in an increase in the number of copies and in an increased stability of the expression plasmids in the said hosts. For example, such restriction sites may correspond to sequences such as 5′-GGCCNNNNNGGCC-3′ SEQ ID NO: 19 (SfiI) or 5′-GCGGCCGC-3′ (NotI) in so far as these sites are extremely rare and generally absent from an expression vector.
After construction of such vectors or expression cassette, the latter are introduced into the host cells selected according to the conventional techniques described in the literature. In this respect, any method permitting the introduction of a foreign DNA into a cell can be used. This may be especially transformation, electroporation, conjugation, or any other technique known to persons skilled in the art. As an example of yeast-type hosts, the various strains of Kluyveromyces used were transformed by treating the whole cells in the presence of lithium acetate and polyethylene glycol, according to the technique described by Ito et al. [J. Bacteriol. 153 (1983) 163]. The transformation technique described by Durrens et al. [Curr. Genet. 18 (1990) 7] using ethylene glycol and dimethyl sulphoxide was also used. It is also possible to transform the yeasts by electroporation, according to the method described by Karube et al. [FEBS Letters 182 (1985) 90]. An alternative procedure is also described in detail in the examples below.
After selection of the transformed cells, the cells expressing the said polypeptides are inoculated and the recovery of the said polypeptides can be carried out, either during the cell growth for the “continuous” processes, or at the end of growth for the “batch” cultures. The polypeptides which are the subject of the present invention are then purified from the culture supernatant for their molecular, pharmacokinetic and biological characterization.
A preferred expression system for the polypeptides of the invention consists in using yeasts of the genus Kluyveromyces as host cell, transformed by certain vectors derived from the extrachromosomal replicon pKD 1 originally isolated from K. marxianus var. drosophilarum. These yeasts, and in particular K. lactis and K. fragilis are generally capable of stably replicating the said vectors and possess, in addition, the advantage of being included in the list of G.R.A.S. (“Generally Recognized As Safe”) organisms. Favoured yeasts are preferably industrial yeasts of the genus Kluyveromyces which are capable of stably replicating the said plasmids derived from the plasmid pKD1 and in which has been inserted a selectable marker as well as an expression cassette permitting the secretion, at high levels, of the polypeptides of the invention.
The present invention also relates to the nucleotide sequences encoding the chimeric polypeptides described above, as well as the eukaryotic or prokaryotic recombinant cells comprising such sequences.
The present invention also relates to the application, as medicinal products, of the polypeptides according to the present invention. More particularly, the subject of the invention is any pharmaceutical composition comprising one or more polypeptides or nucleotide sequences as described above. The nucleotide sequences can indeed be used in gene therapy.
The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and non-limiting.
The representations of the plasmids indicated in the following figures are not plotted to scale and only the restriction sites important for the understanding of the clonings carried out have been indicated.
a) to 2(c), together, comprise an example of a nucleotide sequence (SEQ ID NO:1) and an amino acid sequence (SEQ ID NO:2) of a HindIII restriction fragment encoding a chimeric protein of the prepro-HSA-PEPTIDE type. The black arrows indicate the end of the “pre” and “pro” regions of HSA. The MstII restriction site is underligned and the codon specifying the termination of translation is in bold characters.
In
a) to 11(d) together comprise the nucleotide sequence (SEQ ID NO:15) and amino acid sequence (SEQ ID NO:16) of the HindIII restriction fragment of the plasmid pYG1301 (chimera G.CSF-Gly4-HSA). The black arrows indicate the end of the “pre” and “pro” regions of HSA. The ApaI, SstI (SacI) and MstII restriction sites are underlined. The G.CSF (174 residues) and HSA (585 residues) domains are separated by the synthetic linker GGGG. The numbering of the amino acids corresponds to the mature chimeric protein G.CSF-Gly4-SAH (763 residues). The nucleotide sequence between the translation termination codon and the HindIII site comes from the HSA complementary DNA (cDNA) as described in Patent Application EP 361 991.
B, immunological characterization of the material secreted after using primary antibodies directed against the human G-CSF: same legend as in A.
The methods conventionally used in molecular biology, such as the preparative extractions of plasmid DNA, the centrifugation of plasmid DNA in caesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, extractions of proteins with phenol or phenol-chloroform, DNA precipitation in saline medium with ethanol or isopropanol, transformation in Escherichia coli, and the like are well known to persons skilled in the art and are widely described in the literature [Maniatis T. et al., “Molecular Cloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “Current Protocols in Molecular Biology”, John Wiley & Sons, New York, 1987].
The restriction enzymes were provided by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used according to the recommendations of the suppliers.
The pBR322 and pUC type plasmids and the phages of the M13 series are of commercial origin (Bethesda Research Laboratories).
For the ligations, the DNA fragments are separated according to their size by electrophoresis on agarose or acrylamide gels, extracted with phenol or with a phenol/chloroform mixture, precipitated with ethanol and then incubated in the presence of phage T4 DNA ligase (Biolabs) according to the recommendations of the manufacturer.
The filling of the protruding 5′ ends is carried out by the Klenow fragment of DNA polymerase I of E. coli (Biolabs) according to the specifications of the supplier. The destruction of the protruding 3′ ends is carried out in the presence of phage T4 DNA polymerase (Biolabs) used according to the recommendations of the manufacturer. The destruction of the protruding 5′ ends is carried out by a controlled treatment with S1 nuclease.
Site-directed mutagenesis in vitro with synthetic oligodeoxynucleotides is carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 (1985) 8749–8764] using the kit distributed by Amersham.
The enzymatic amplification of DNA fragments by the so-called PCR technique [Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230 (1985) 1350–1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 335–350] is carried out using a “DNA thermal cycler” (Perkin Elmer Cetus) according to the specifications of the manufacturer.
The verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 5463–5467] using the kit distributed by Amersham.
The transformations of K. lactis with DNA from the plasmids for expression of the proteins of the present invention are carried out by any technique known to persons skilled in the art, and of which an example is given in the text.
Except where otherwise stated, the bacterial strains used are E. coli MC1060 (lacIPOZYA, X74, galU, galK, strAr), or E. coli TG1 (lac, proA,B, supE, thi, hsdD5/FtraD36, proA+B+, lacIq, lacZ, M15).
The yeast strains used belong to the budding yeasts and more particularly to yeasts of the genus Kluyveromyces. The K. lactis MW98-8C (a, uraA, arg, lys, K+, pKD1°) and K. lactis CBS 293.91 strain were particularly used; a sample of the MW98-8C strain was deposited on Sep. 16, 1988 at Centraalbureau voor Schimmelkulturen (CBS) at Barn (the Netherlands) where it was registered under the number CBS 579.88.
A bacterial strain (E. coli) transformed with the plasmid pET-8c52K was deposited on Apr. 17, 1990 with the American Type Culture Collection under the number ATCC 68306.
The yeast strains transformed with the expression plasmids encoding the proteins of the present invention are cultured in erlenmeyers or in 21 pilot fermenters (SETRIC, France) at 28° C. in rich medium (YPD: 1% yeast extract, 2% Bactopeptone, 2% glucose; or YPL: 1% yeast extract, 2% Bactopeptone, 2% lactose) with constant stirring.
The plasmid pYG404 is described in Patent Application EP 361 991. This plasmid contains a HindIII restriction fragment encoding the prepro-HSA gene preceded by the 21 nucleotides naturally present immediately upstream of the initiator ATG for translation of the PGK gene of S. cerevisiae. The nucleotide sequence of this restriction fragment is included in that of
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including the biologically active peptide and the mature form of HSA or one of its molecular variants. These hybrid genes are preferably bordered in 5′ of the translational initiator ATG and in 3′ of the translational stop codon by HindIII restriction sites and encode chimeric proteins of the PEPTIDE-HSA type (
The combined techniques of site-directed mutagenesis and PCR amplification described in Examples 1 and 2 make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between the mature form of HSA, or one of its molecular variants, and a biologically active peptide coupled to the N- and C-terminal ends of HSA. These hybrid genes are preferably bordered in 5′ of the translational initiator ATG and in 3′ of the translational stop codon by HindIII restriction sites and encode chimeric proteins of the PEPTIDE-HSA-PEPTIDE type (
The chimeric proteins of the preceding examples can be expressed in yeasts using functional, regulatable or constitutive promoters such as, for example, those present in the plasmids pYG105 (LAC4 promoter of Kluyveromyces lactis), pYG106 (PGK promoter of Saccharomyces cerevisiae), pYG536 (PHO5 promoter of S. cerevisiae), or hybrid promoters such as those described in Patent Application EP 361 991. The plasmids pYG105 and pYG106 are particularly useful here because they permit the expression of the genes encoded by the HindIII restriction fragments as described in the preceding examples and cloned into the HindIII site and in the productive orientation (defined as the orientation which places the “prepro” region of albumin proximally relative to the promoter for transcription), using promoters which are functional in K. lactis, regulatable (pYG105) or constitutive (pYG106). The plasmid pYG105 corresponds to the plasmid pKan707 described in Patent Application EP 361 991 in which the HindIII restriction site which is unique and localized in the gene for resistance to geneticin (G418) has been destroyed by site-directed mutagenesis while preserving an unchanged protein (oligodeoxynucleotide 5′-GAAATGCATAAGCTCTTGCCATTCTCACCG-3′)(SEQ ID NO:21). The SalI-SacI fragment encoding the URA3 gene of the mutated plasmid was then replaced with a SalI-SacI restriction fragment containing an expression cassette consisting of the LAC4 promoter of K. lactis (in the form of a SalI-HindIII fragment) and the terminator of the PGK gene of S. cerevisiae (in the form of a HindIII-SacI fragment). The plasmid pYG105 is mitotically very stable in the Kluyveromyces yeasts and a restriction map thereof is given in
The transformation of the yeasts belonging to the genus Kluyveromyces, and in particular the strains MW98-8C and CBS 293.91 of K. lactis is carried out for example by the technique for treating whole cells with lithium acetate [Ito H. et al., J. Bacteriol. 153 (1983) 163–168], adapted as follows. The growth of the cells is carried out at 28° C. in 50 ml of YPD medium, with stirring and up to an optical density of 600 nm (OD600) of between 0.6 and 0.8; the cells are harvested by centrifugation at low speed, washed in a sterile solution of TE (10 mM Tris HCl pH 7.4; 1 mM EDTA), resuspended in 3–4 ml of lithium acetate (0.1M in TE) in order to obtain a cellular density of about 2×108 cells/ml, and then incubated at 30° C. for 1 hour with moderate stirring. Aliquots of 0.1 ml of the resulting suspension of competent cells are incubated at 30° C. for 1 hour in the presence of DNA and at a final concentration of 35% polyethylene glycol (PEG4000, Sigma). After a heat shock of 5 minutes at 42° C., the cells are washed twice, resuspended in 0.2 ml of sterile water and incubated for 16 hours at 28° C. in 2 ml of YPD medium in order to permit the phenotypic expression of the gene for resistance to G418 expressed under the control of the Pkl promoter (cf. EP 361 991); 200 μl of the cellular suspension are then plated on selective YPD dishes (G418, 200 μg/ml). The dishes are incubated at 28° C. and the transformants appear after 2 to 3 days of cell growth.
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins. Few clones, corresponding to the strain CBS 293.91 or MW98-8C transformed by the plasmids for expression of the chimeras between HSA and the biologically active part, are incubated in YPD or YPL medium at 28° C. The cellular supernatants are recovered by centrifugation when the cells reach the stationary growth phase, optionally concentrated 10 times by precipitation for 30 minutes at −20° C. in a final concentration of 60% ethanol, and then tested after electrophoresis on an 8.5% SDS-PAGE gel, either directly by staining the gel with coomassie blue, or after immunoblotting using primary antibodies directed against the biologically active part or a rabbit polyclonal serum directed against HSA. During the experiments for immunological detection, the nitrocellulose filter is first incubated in the presence of specific primary antibodies, washed several times, incubated in the presence of goat antibodies directed against the primary antibodies, and then incubated in the presence of an avidin-peroxidase complex using the “ABC kit” distributed by Vectastain (Biosys S. A., Compiegne, France). The immunological reaction is then revealed by the addition of 3,3′-diamino benzidine tetrahydrochloride (Prolabo) in the presence of hydrogen peroxide, according to the recommendations of the manufacturer.
E.7.1. Fragments Antagonizing the Binding of vWF to the Platelets
E.7.1.1. Thr470-Val713 Residues of vWF
The plasmid pET-8c52K contains a fragment of the vWF cDNA encoding residues 445 to 733 of human vWF and therefore includes several crucial determinants of the interaction between vWF and the platelets on the one hand, and certain elements of the basal membrane and the sub-endothelial tissue on the other, and especially the peptides G10 and D5 which antagonize the interaction between vWF and GPlb [Mori H. et al., J. Biol. Chem. 263 (1988) 17901–17904]. This peptide sequence is identical to the corresponding sequence described by Titani et al. [Biochemistry 25, (1986) 3171–3184]. The amplification of these genetic determinants can be carried out using the plasmid pET-8c52K, for example by the PCR amplification technique, using as primer oligodeoxynucleotides encoding contiguous residues localized on either side of the sequence to be amplified. The amplified fragments are then cloned into vectors of the M13 type for their verification by sequencing using either the universal primers situated on either side of the multiple cloning site, or oligodeoxynucleotides specific for the amplified region of the vWF gene of which the sequence of several isomorphs is known [Sadler J. E. et al., Proc. Natl. Acad. Sci. 82 (1985) 6394–6398; Verweij C. L. et al., EMBO J. 5 (1986) 1839–1847; Shelton-Inloe B. B. et al., Biochemistry 25 (1986) 3164–3171; Bonthron D. et al., Nucleic Acids Res. 17 (1986) 7125–7127]. Thus, the PCR amplification of the plasmid pET-8c52K with the oligodeoxynucleotides 5′-CCCGGGATCCCTTAGGCTTAACCTGTGAAGCCTGC-3′ (SEQ ID NO:22) (Sql969, the MstI site is underlined) and 5′-CCCGGGATCCAAGCTTAGACTTGTGCCATGTCG-3′ (SEQ ID NO:23) (Sq2029, the HindIII site is underlined) generates an MstII-HindIII restriction fragment including the Thr470 to Val713 residues of vWF (
E.7.1.2. Molecular Variants:
In another embodiment, the binding site of vWF is a peptide including the Thr470 to Asp498 residues of the mature vWF. This sequence including the peptide G10 (Cys474-Pro488) described by Mori et al. [J. Biol. Chem. 263 (1988) 17901–17904] and capable of antagonizing the interaction of human vWF with the GP1b of the human platelets. The sequence corresponding to the peptide G10 is first included in an MstII-HindIII restriction fragment (
(the sequence encoding the peptide G10 is in bold characters). The ligation of this fragment to the HindIII-MstII restriction fragment corresponding to the entire gene encoding HSA, with the exception of the three C-terminalmost amino acids (cf.
In another embodiment, the site for binding of vWF to GPlb is directly designed with the aid of synthetic oligodeoxynucleotides, and for example the oligodeoxynucleotides
These oligodeoxynucleotides form, by pairing, a MstII-BglII restriction fragment including the MstII-HindIII fragment (
Useful variants of the plasmid pET-8c52K are deleted by site-directed mutagenesis between the peptides G10 and G5, for example sites for binding to collagen, and/or to heparin, and/or to botrocetin, and/or to sulphatides and/or to ristocetin. One example is the plasmid pMMB9 deleted by site-directed mutagenesis between the residues Cys509 and 11e662. The PCR amplification of this plasmid with the oligodeoxynucleotides Sq1969 and Sq2029 generates an MstII-HindIII restriction fragment (
In other embodiments, the use of combined techniques of site-directed mutagenesis and PCR amplification makes it possible to generate at will variants of the MstII-HindIII restriction fragment of panel A of
In other useful variants of the plasmid pET-8c52K, mutations are introduced, for example by site-directed mutagenesis, in order to replace or suppress all or part of the set of cysteines present at positions 471, 474, 509 and 695 of the human vWF. Specific examples are the plasmids p5E and p7E in which the cysteins present at positions 471 and 474, on the one hand, and at positions 471, 474, 509 and 695, on the other hand, have been respectively replaced by glycine residues. The PCR amplification of these plasmids with the oligodeoxynucleotides Sq2149 (5′-CCCGGGATCCCTTAGGCTTAACCGGTGAAGCCGGC-3′ (SEQ ID NO:28), the MstII site is underlined) and Sq2029 makes it possible to generate MstII-HindIII restriction fragments including the Thr470 to Val713 residues of the natural vWF with the exception that at least the cystein residues at positions 471 and 474 were mutated to glycine residues. The ligation of these fragments to the HindIII-MstI restriction fragment corresponding to the entire gene encoding HSA with the exception of the three C-terminalmost amino acids (cf.
Other particularly useful mutations affect at least one residue involved in vWF-associated type IIB pathologies (increase in the intrinsic affinity of vWF for GP1b), such as the residues Arg543, Arg545, Trp550, Val551, Val553, Pro574 or Arg578 for example. The genetic recombination techniques in vitro also make it possible to introduce at will one or more additional residues into the sequence of vWF and for example a supernumerary methionine between positions Asp539 and Glu542.
E.7.2. Fragments Antagonizing the Binding of vWF to the Sub-Endothelium
In a specific embodiment, the sites for binding of vWF to the components of the sub-endothelial tissue, and for example collagen, are generated by PCR amplification of the plasmid pET-8c52K, for example with the oligodeoxynucleotides
the HindIII site is underlined), which generates an MstII-HindIII restriction fragment encoding the Cys509 to Cys695 residues of the natural vWF. Deletion molecular variants or modified variants are also generated which contain any desired combination between the sites for binding of vWF to the sulphatides and/or to botrocetin and/or to heparin and/or to collagen and/or any residue responsible for a modification of the affinity of vWF for GP1b (vWF-associated type II pathologies). In another embodiment, the domain capable of binding to collagen may also come from the vWF fragment which is between the residues 911 and 1114 and described by Pareti et al. [J. Biol. Chem. (1987) 262: 13835–13841]. The ligation of these fragments to the HindIII-MstII restriction fragment corresponding to the entire gene encoding HSA with the exception of the three C-terminalmost amino acids (cf.
E.7.3. Purification and Molecular Characterization of the Chimeras Between HSA and vWF
The chimeras present in the culture supernatants corresponding to the CBS 293.91 strain transformed, for example with the expression plasmids according to Examples E.7. 1. and E.7.2., are characterized in a first instance by means of antibodies specific for the HSA part and for the vWF part. The results of
E.8.1. Constructs
A fragment corresponding to the amino-terminal fragment of urokinase (ATF: EGF-like domain+ringle domain) can be obtained from the corresponding messenger RNA of cells of certain human carcinoma, for example using the RT-PCR kit distributed by Pharmacia. An MstII-HindIII restriction fragment including the ATF of human urokinase is given in
E.8.2. Secretion of the Hybrids
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins HSA-UK. A few clones corresponding to the strain K. lactis CBS 293.91, which is transformed with the expression plasmids according to Example E.9.1., are incubated in selective complete liquid medium at 28° C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin or against human urokinase. The results of
E.8.3 Purification of the Chimeras Between HSA and Urokinase
After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.8.1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa. The concentrate obtained is then adjusted to 50 mM Tris-HCl starting with a stock solution of 1M Tris-HCl (pH 7), and then loaded in 20 ml fractions onto an anion-exchange column (3 ml) (D-Zephyr, Sepracor) equilibrated in the same buffer. The chimeric protein (HSA-UK1→146 or HSA-UK1→135) is then eluted from the column by a gradient (0 to 1M) of NaCl. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCl solution (pH 6) and reloaded onto a D-Zephyr column equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization of their biological activity and especially with respect to their ability to displace urokinase from its cellular receptor.
E.9.1. Constructs
E.9.1.1. Coupling at the C-terminus of HSA.
An MstII-HindIII restriction fragment including the mature form of human G-CSF is generated, for example according to the following strategy: a KpnI-HindIII restriction fragment is first obtained by the enzymatic PCR amplification technique using the oligodeoxynucleotides Sq2291 (5′-CAAGGATCC-AAGCTTCAGGGCTGCGCAAGGTGGCGTAG-3′ (SEQ ID NO:31), the HindIII site is underlined) and Sq2292 (5′-CGGGGTACCTTAGGCTTAACCCCCCTG-GGCCCTGCCAGC-3′ (SEQ ID NO:32), the KpnI site is underlined) as primer on the plasmid BBG13 serving as template. The plasmid BBG13 contains the gene encoding the B form (174 amino acids) of mature human G-CSF, which is obtained from British Bio-technology Limited, Oxford, England. The enzymatic amplification product of about 550 nucleotides is then digested with the restriction enzymes KpnI and HindIII and cloned into the vector pUC19 cut with the same enzymes, which generates the recombinant plasmid pYG1255. This plasmid is the source of an MstII-HindIII restriction fragment which makes it possible to fuse G-CSF immediately downstream of HSA (chimera HSA-G.CSF) and whose nucleotide sequence is given in
It may also be desirable to insert a peptide linker between the HSA part and G-CSF, for example in order to permit a better functional presentation of the transducing part. An MstII-HindIII restriction fragment is for example generated by substitution of the MstII-ApaI fragment of the plasmid pYG1255 by the oligodeoxynucleotides Sq2742 (5′-TTAGGCTTAGGTGGTGGCGGT-ACCCCCCTGGGCC-3′ (SEQ ID NO:33), the codons encoding the glycine residues of this particular linker are underlined) and Sq2741 (5′-CAGGGGGGTACCGCCACCACCTAAGCC-3′) (SEQ ID NO:34) which form, by pairing, an MstII-ApaI fragment. The plasmid thus generated therefore contains an MstII-HindIII restriction fragment whose sequence is identical to that of
The ligation of the HindIII-MstII fragment of the plasmid pYG404 to the MstII-HindIII fragment of the plasmid pYG1255 makes it possible to generate the HindIII fragment of the plasmid pYG1259 which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule (HSA-G.CSF).
An identical HindIII restriction fragment, with the exception of the MstII-ApaI fragment, may also be easily generated and which encodes a chimeric protein in which the B form of the mature G-CSF is positioned by genetic coupling in translational phase at the C-terminus of the HSA molecule and a specific peptide linker. For example, this linker consists of 4 glycine residues in the HindIII fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF).
The HindIII restriction fragment of the plasmid pYG1259 is cloned in the productive orientation and into the HindIII restriction site of the expression plasmid pYG105, which generates the expression plasmid pYG1266 (HSA-G.CSF). In another exemplification, the cloning of the HindIII restriction fragment of the plasmid pYG1259 in the productive orientation and into the HindIII site of the plasmid pYG106 generates the plasmid pYG1267. The plasmids pYG1266 and pYG1267 are mutually isogenic with the exception of the SalI-HindIII restriction fragment encoding the LAC4 promoter of K. lactis (plasmid pYG1266) or the PGK promoter of S. cerevisiae (plasmid pYG1267).
In another exemplification, the cloning in the productive orientation of the HindIII restriction fragment of the plasmid pYG1336 (chimera HSA-Gly4-G.CSF) into the HindIII site of the plasmids pYG105 (LAC4) and pYG106 (PGK) generates the expression plasmids pYG1351 and pYG1352 respectively.
E.9.1.2. Coupling at the N-terminus of HSA
In a specific embodiment, the combined techniques of site-directed mutagenesis and PCR amplification make it possible to construct hybrid genes encoding a chimeric protein resulting from the translational coupling between a signal peptide (and for example the prepro region of HSA), a sequence including a gene having a G-CSF activity, and the mature form of HSA or one of its molecular variants (cf. chimera of panel B,
GTGGAGGCGGTGATGCACACAAGAGTGAGGTTGCTCATCGG-3′
the residues underlined (optional) correspond in this particular chimera to a peptide linker composed of 4 glycine residues) makes it possible, by site-directed mutagenesis, to put in translational phase the mature form of the human G-CSF of the plasmid BBG 13 immediately upstream of the mature form of HSA, which generates the intermediate plasmid A. Likewise, the use of the oligodeoxynucleotide
GTTCGACGAAACACACCCCTGGAATAAGCCGAGCT-3′
(non-coding strand), the nucleotides complementary to the nucleotides encoding the first N-terminal residues of the mature form of the human G-CSF are underlined] makes it possible, by site-directed mutagenesis, to couple in translational reading phase the prepro region of HSA immediately upstream of the mature form of the human G-CSF, which generates the intermediate plasmid B. A HindIII fragment encoding a chimeric protein of the PEPTIDE-HSA type (cf.
E.9.2. Secretion of the Hybrids.
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric proteins between HSA and G-CSF. A few clones corresponding to the strain K. lactis CBS 293.91 transformed with the plasmids pYG1266 or pYG1267 (HSA-G.CSF), pYG1302 or pYG1303 (G.CSF-Gly4-HSA) or alternatively pYG1351 or pYG 1352 (HSA-Gly4-G.CSF) are incubated in selective complete liquid medium at 28° C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining the gel with coomassie blue, or after immunoblotting using as primary antibodies rabbit polyclonal antibodies directed against the human G-CSF or a rabbit polyclonal serum directed against human albumin. The results of
E.9.3. Purification and Molecular Characterization of the Chimeras Between HSA and G-CSF.
After centrifugation of a culture of the CBS 293.91 strain transformed with the expression plasmids according to Example E.9. 1., the culture supernatant is passed through a 0.22 mm filter (Millipore) and then concentrated by ultrafiltration (Amicon) using a membrane whose discrimination threshold is situated at 30 kDa. The concentrate obtained is then adjusted to 50 mM Tris-HCl from a 1M stock solution of Tris-HCl (pH 6), and then loaded in 20 ml fractions onto an ion-exchange column (5 ml) (Q Fast Flow, Pharmacia) equilibrated in the same buffer. The chimeric protein is then eluted from the column by a gradient (0 to 1M) of NaCl. The fractions containing the chimeric protein are then pooled and dialysed against a 50 mM Tris-HCl solution (pH 6) and reloaded onto a Q Fast Flow column (1 ml) equilibrated in the same buffer. After elution of the column, the fractions containing the protein are pooled, dialysed against water and freeze-dried before characterization: for example, the sequencing (Applied Biosystem) of the protein HSA-G.CSF secreted by the yeast CBS 293.91 gives the N-terminal sequence expected for HSA (Asp-Ala-His . . . ), demonstrating a correct maturation of the chimera immediately at the C-terminus of the doublet of residues Arg-Arg of the “pro” region of HSA (
E.10.1. Constructs
An Fv′ fragment can be constructed by genetic engineering techniques, and which encodes the variable fragments of the heavy and light chains of an immunoglobulin (Ig), linked to each other by a linker peptide [Bird et al., Science (1988) 242: 423; Huston et al., (1988) Proc. Natl. Acad. Sci. 85: 5879]. Schematically, the variable regions (about 120 residues) of the heavy and light chains of a given Ig are cloned from the messenger RNA of the corresponding hybridoma, for example using the RT-PCR kit distributed by Pharmacia (Mouse ScFv module). In a second stage, the variable regions are genetically coupled by genetic engineering via a synthetic linkage peptide and for example the linker (GGGGS)x3. An MstII-HindIII restriction fragment including the Fv′ fragment of an immunoglobulin secreted by a murine hybridoma is given in
E.10.2. Secretion of the Hybrids
After selection on rich medium supplemented with G418, the recombinant clones are tested for their capacity to secrete the mature form of the chimeric protein HSA-Fv′. A few clones corresponding to the strain K. lactis CBS 293.91 transformed with the plasmids pYG1383 or pYG1384 (HSA-Fv′) are incubated in selective complete liquid medium at 28° C. The cellular supernatants are then tested after electrophoresis on an 8.5% acrylamide gel, either directly by staining of the gel with coomassie blue, or after immunoblotting using as primary antibodies a rabbit polyclonal serum directed against human albumin, or directly incubated with biotinylated antibodies directed against the immunoglobulins of murine origin. The results of
E.11.1. Biological Activity In Vitro.
E.11.1. Chimeras Between HSA and vWF.
The antagonistic activity of the products is determined by measuring the dose-dependent inhibition of the agglutination of human platelets fixed with paraformaldehyde according to the method described by Prior et al. [Bio/Technology (1992) 10: 66]. The measurements are carried out in an aggregameter (PAP-4, Bio Data, Horsham, Pa., U.S.A.) which records the variations over time of the optical transmission, with stirring, at 37° C. in the presence of vWF, of botrocetin (8.2 mg/ml) and of the test product at various dilutions (concentrations). For each measurement, 400 ml (8×107 platelets) of a suspension of human platelets stabilized with paraformaldehyde (0.5%, and then resuspended in [NaCl (137 mM); MgCl2 (1 mM); NaH2 PO4 (0.36 mM); NaHCO3 (10 mM); KCl (2.7 mM); glucose (5.6 mM); HSA (3.5 mg/ml); HEPES buffer (10 mM, pH 7.35)] are preincubated at 37° C. in the cylindrical tank (8.75×50 mm, Wellcome Distriwell, 159 rue Nationale, Paris) of the aggregameter for 4 min and are then supplemented with 30 ml of the solution of the test product at various dilutions in apyrogenic formulation vehicle [mannitol (50 g/l); citric acid (192 mg/l); L-lysine monohydrochloride (182.6 mg/l); NaCl (88 mg/l); pH adjusted to 3.5 by addition of NaOH (IM)], or formulation vehicle alone (control assay). The resulting suspension is then incubated for 1 min at 37° C. and 12.5 ml of human vWF [American Bioproducts, Parsippany, N.J., U.S.A.; 11% von Willebrand activity measured according to the recommendations for the use of PAP-4 (Platelet Aggregation Profiler®) with the aid of platelets fixed with formaldehyde (2×105 platelets/ml), human plasma containing 0 to 100% vWF and ristocetin (10 mg/ml, cf. p. 36–45: vW Program™] are added and incubated at 37° C. for 1 min before adding 12.5 ml of botrocetin solution [purified from freeze-dried venom of Bothrops jararaca (Sigma) according to the procedure described by Sugimoto et al., Biochemistry (1991) 266: 18172]. The recording of the reading of the transmission as a function of time is then carried out for 2 min with stirring by means of a magnetic bar (Wellcome Distriwell) placed in the tank and with a magnetic stirring of 1,100 rpm provided by the aggregameter. The mean variation of the optical transmission (n3 5 for each dilution) over time is therefore a measurement of the platelet agglutination due to the presence of vWF and botrocetin, in the absence or in the presence of variable concentrations of the test product. From such recordings, the % inhibition of the platelet agglutination due to each concentration of product is then determined and the straight line giving the % inhibition as a function of the reciprocal of the product dilution in log-log scale is plotted. The IC50 (or concentration of product causing 50% inhibition of the agglutination) is then determined on this straight line. The table of
E.11.1.2. Chimeras between HSA and G-CSF
The purified chimeras are tested for their capacity to permit the in vitro proliferation of the IL3-dependant murine line NFS60, by measuring the incorporation of tritiated thymidine essentially according to the procedure described by Tsuchiya et al. [Proc. Natl. Acad. Sci. (1986) 83 7633]. For each chimera, the measurements are carried out between 3 and 6 times in a three-point test (three dilutions of the product) in a zone or the relation between the quantity of active product and incorporation of labelled thymidine (Amersham) is linear. In each microtitre plate, the activity of a reference product consisting of recombinant human G-CSF expressed in mammalian cells is also systematically incorporated. The results of
E.11.2. Biological Activity in Vivo
The activity of stimulation of the HSA-G-CSF chimeras on granulopoiesis in vivo is tested after subcutaneous injection in rats (Sprague-Dawley/CD, 250–300 g, 8–9 weeks) and compared to that of the reference G-CSF expressed using mammalian cells. Each product, tested at the rate of 7 animals, is injected subcutaneously into the dorso-scapular region at the rate of 100 ml for 7 consecutive days, (D1–D7). 500 ml of blood are collected on days D-6, D2 (before the 2nd injection). D5 (before the 5th injection) and D8, and a blood count is performed. In this test, the specific activity (neutropoiesis units/mole injected) of the chimera HSA-G.CSF (pYG1266) is identical to that of the reference G-CSF (
| Number | Date | Country | Kind |
|---|---|---|---|
| 92 01064 | Jan 1992 | FR | national |
This application is a DIV of 09/984,186 Oct. 29, 2001, now U.S. Pat. No. 6,686,179, which is a CON of 09/258,532 Feb. 26, 1999, now abandoned, which is a DIV of 08/797,689 Jan. 31, 1997, now U.S. Pat. No. 5,876,969, which is a CON of 08/256,927 Jul. 28, 1994, now abandoned.
| Number | Name | Date | Kind |
|---|---|---|---|
| 4264731 | Shine | Apr 1981 | A |
| 4283489 | Goodman et al. | Aug 1981 | A |
| 4336248 | Bonhard et al. | Jun 1982 | A |
| 4342832 | Goeddel et al. | Aug 1982 | A |
| 4363877 | Goodman et al. | Dec 1982 | A |
| 4366246 | Riggs | Dec 1982 | A |
| 4397840 | Takezawa et al. | Aug 1983 | A |
| 4407948 | Goodman et al. | Oct 1983 | A |
| 4440859 | Rutter et al. | Apr 1984 | A |
| 4447538 | Goodman et al. | May 1984 | A |
| 4450103 | Konrad et al. | May 1984 | A |
| 4462940 | Hanisch et al. | Jul 1984 | A |
| 4492684 | Goosen et al. | Jan 1985 | A |
| 4499188 | Konrad et al. | Feb 1985 | A |
| 4652525 | Rutter et al. | Mar 1987 | A |
| 4670393 | Seeburg | Jun 1987 | A |
| 4751180 | Cousens et al. | Jun 1988 | A |
| 4765980 | DePrince et al. | Aug 1988 | A |
| 4775622 | Hitzeman et al. | Oct 1988 | A |
| 4778879 | Mertelsmann et al. | Oct 1988 | A |
| 4792602 | Narang et al. | Dec 1988 | A |
| 4801575 | Pardridge | Jan 1989 | A |
| 4835260 | Shoemaker | May 1989 | A |
| 4840934 | Anderson | Jun 1989 | A |
| 4898830 | Goeddel et al. | Feb 1990 | A |
| 4908433 | Mertelsmann et al. | Mar 1990 | A |
| 4908434 | Mertelsmann et al. | Mar 1990 | A |
| 4914026 | Brake et al. | Apr 1990 | A |
| 4914027 | Knapp et al. | Apr 1990 | A |
| 4916212 | Markussen et al. | Apr 1990 | A |
| 4925919 | Mertelsmann et al. | May 1990 | A |
| 4929442 | Powell | May 1990 | A |
| 4959314 | Mark et al. | Sep 1990 | A |
| 4970300 | Fulton et al. | Nov 1990 | A |
| 4999339 | Paradise et al. | Mar 1991 | A |
| 5002764 | Peets et al. | Mar 1991 | A |
| 5010003 | Chang et al. | Apr 1991 | A |
| 5015575 | Brake et al. | May 1991 | A |
| 5028422 | Tanner et al. | Jul 1991 | A |
| 5045312 | Aston et al. | Sep 1991 | A |
| 5053389 | Balschmidt et al. | Oct 1991 | A |
| 5061488 | Wiltrout et al. | Oct 1991 | A |
| 5066489 | Paradise et al. | Nov 1991 | A |
| 5071872 | Witiak et al. | Dec 1991 | A |
| 5073627 | Curtis et al. | Dec 1991 | A |
| 5096707 | Wiltrout et al. | Mar 1992 | A |
| 5096885 | Pearlman et al. | Mar 1992 | A |
| 5100784 | Latta et al. | Mar 1992 | A |
| 5102872 | Singh et al. | Apr 1992 | A |
| 5106954 | Fibi et al. | Apr 1992 | A |
| 5116944 | Sivam et al. | May 1992 | A |
| 5118666 | Habener | Jun 1992 | A |
| 5120712 | Habener | Jun 1992 | A |
| 5126129 | Wiltrout et al. | Jun 1992 | A |
| 5128126 | Boniver | Jul 1992 | A |
| 5187261 | Latta et al. | Feb 1993 | A |
| 5208018 | Gough | May 1993 | A |
| 5219565 | Brandely et al. | Jun 1993 | A |
| 5229109 | Grimm et al. | Jul 1993 | A |
| 5230886 | Treon et al. | Jul 1993 | A |
| 5256410 | Tanner et al. | Oct 1993 | A |
| 5260202 | Clarke et al. | Nov 1993 | A |
| 5272070 | Lehrman et al. | Dec 1993 | A |
| 5302697 | Goodey et al. | Apr 1994 | A |
| 5304473 | Belagaje et al. | Apr 1994 | A |
| 5322930 | Tarnowski et al. | Jun 1994 | A |
| 5330971 | Wells et al. | Jul 1994 | A |
| 5336603 | Capon et al. | Aug 1994 | A |
| 5358709 | Tursz et al. | Oct 1994 | A |
| 5380712 | Ballance et al. | Jan 1995 | A |
| 5395922 | Bjørn et al. | Mar 1995 | A |
| 5409815 | Nakagawa et al. | Apr 1995 | A |
| 5424199 | Goeddel et al. | Jun 1995 | A |
| 5432082 | Galeotti et al. | Jul 1995 | A |
| 5459031 | Blumen et al. | Oct 1995 | A |
| 5460811 | Goeddel et al. | Oct 1995 | A |
| 5460954 | Lee et al. | Oct 1995 | A |
| 5503993 | Hayasuke et al. | Apr 1996 | A |
| 5508031 | Zimmerman et al. | Apr 1996 | A |
| 5512549 | Chen et al. | Apr 1996 | A |
| 5521086 | Scott et al. | May 1996 | A |
| 5545618 | Buckley et al. | Aug 1996 | A |
| 5567677 | Castensson et al. | Oct 1996 | A |
| 5574008 | Johnson et al. | Nov 1996 | A |
| 5582822 | Brandely et al. | Dec 1996 | A |
| 5602232 | Reichert et al. | Feb 1997 | A |
| 5612196 | Becquart et al. | Mar 1997 | A |
| 5614492 | Habener | Mar 1997 | A |
| 5618676 | Hitzeman et al. | Apr 1997 | A |
| 5618698 | Lin | Apr 1997 | A |
| 5625041 | Johnson et al. | Apr 1997 | A |
| 5629286 | Brewitt | May 1997 | A |
| 5633352 | Dalboge et al. | May 1997 | A |
| 5637504 | Hinchliffe et al. | Jun 1997 | A |
| 5639642 | Kjeldsen et al. | Jun 1997 | A |
| 5641663 | Garvin et al. | Jun 1997 | A |
| 5646012 | Fleer et al. | Jul 1997 | A |
| 5646113 | Attie et al. | Jul 1997 | A |
| 5658568 | Bagshawe et al. | Aug 1997 | A |
| 5665863 | Yeh et al. | Sep 1997 | A |
| 5667986 | Goodey et al. | Sep 1997 | A |
| 5679777 | Anderson et al. | Oct 1997 | A |
| 5702717 | Cha et al. | Dec 1997 | A |
| 5705363 | Imakawa et al. | Jan 1998 | A |
| 5714377 | Tanner et al. | Feb 1998 | A |
| 5726038 | Christiansen et al. | Mar 1998 | A |
| 5728553 | Goodey et al. | Mar 1998 | A |
| 5728707 | Wehrmann | Mar 1998 | A |
| 5739007 | Kingsman et al. | Apr 1998 | A |
| 5741815 | Lai | Apr 1998 | A |
| 5763394 | O'Connor et al. | Jun 1998 | A |
| 5766620 | Heiber et al. | Jun 1998 | A |
| 5766883 | Ballance et al. | Jun 1998 | A |
| 5767097 | Tam | Jun 1998 | A |
| 5780021 | Sobel | Jul 1998 | A |
| 5783423 | Wood et al. | Jul 1998 | A |
| 5788964 | Baral et al. | Aug 1998 | A |
| 5795745 | Goeddel et al. | Aug 1998 | A |
| 5795746 | Kjeldsen et al. | Aug 1998 | A |
| 5795777 | Taniguchi et al. | Aug 1998 | A |
| 5801190 | Hudkins et al. | Sep 1998 | A |
| 5824330 | Mertelsmann et al. | Oct 1998 | A |
| 5830452 | Bauer et al. | Nov 1998 | A |
| 5840542 | Kang et al. | Nov 1998 | A |
| 5844095 | Linsley et al. | Dec 1998 | A |
| 5846774 | Xia | Dec 1998 | A |
| 5847004 | Lai | Dec 1998 | A |
| 5849322 | Ebert | Dec 1998 | A |
| 5854018 | Hitzeman et al. | Dec 1998 | A |
| 5856123 | Hitzeman et al. | Jan 1999 | A |
| 5861406 | Wehrmann | Jan 1999 | A |
| 5863555 | Heiber et al. | Jan 1999 | A |
| 5876969 | Fleer et al. | Mar 1999 | A |
| 5889144 | Alila et al. | Mar 1999 | A |
| 5905143 | Johnson et al. | May 1999 | A |
| 5908830 | Smith et al. | Jun 1999 | A |
| 5912229 | Thim et al. | Jun 1999 | A |
| 5919651 | Hitzeman et al. | Jul 1999 | A |
| 5919815 | Bradley et al. | Jul 1999 | A |
| 5922674 | Anagnostou et al. | Jul 1999 | A |
| 5922761 | Lai | Jul 1999 | A |
| 5932547 | Stevenson et al. | Aug 1999 | A |
| 5939455 | Rephaeli | Aug 1999 | A |
| 5948428 | Lee et al. | Sep 1999 | A |
| 5951996 | Czeizler Zaharia | Sep 1999 | A |
| 5952461 | Kim et al. | Sep 1999 | A |
| 5958909 | Habener | Sep 1999 | A |
| 5965386 | Kerry-Williams et al. | Oct 1999 | A |
| 5968510 | Linsley et al. | Oct 1999 | A |
| 5977071 | Galloway et al. | Nov 1999 | A |
| 5977318 | Linsley et al. | Nov 1999 | A |
| 5981474 | Manning et al. | Nov 1999 | A |
| 5981485 | O'Connor et al. | Nov 1999 | A |
| 5981488 | Hoffmann | Nov 1999 | A |
| 5985850 | Falk et al. | Nov 1999 | A |
| 6004573 | Rathi et al. | Dec 1999 | A |
| 6006753 | Efendic | Dec 1999 | A |
| 6017545 | Modi | Jan 2000 | A |
| 6030961 | Nudelman et al. | Feb 2000 | A |
| 6031004 | Timmins et al. | Feb 2000 | A |
| 6034221 | Berezenko et al. | Mar 2000 | A |
| 6045788 | Smith | Apr 2000 | A |
| 6048724 | Selden et al. | Apr 2000 | A |
| 6054489 | Lorens et al. | Apr 2000 | A |
| 6063373 | Hellstrand et al. | May 2000 | A |
| 6063772 | Tam | May 2000 | A |
| 6069135 | Falk et al. | May 2000 | A |
| 6071923 | Nudelman et al. | Jun 2000 | A |
| 6080877 | Swindell et al. | Jun 2000 | A |
| 6087129 | Newgard et al. | Jul 2000 | A |
| 6110703 | Egel-Mitani et al. | Aug 2000 | A |
| 6110707 | Newgard et al. | Aug 2000 | A |
| 6110891 | Pusztai et al. | Aug 2000 | A |
| 6110955 | Nudelman et al. | Aug 2000 | A |
| 6110970 | Nudelman et al. | Aug 2000 | A |
| 6114146 | Herlitschka et al. | Sep 2000 | A |
| 6117949 | Rathi et al. | Sep 2000 | A |
| 6124495 | Neiss et al. | Sep 2000 | A |
| 6130248 | Nudelman et al. | Oct 2000 | A |
| 6133235 | Galloway et al. | Oct 2000 | A |
| 6149911 | Binz et al. | Nov 2000 | A |
| 6150133 | Mead et al. | Nov 2000 | A |
| 6150337 | Tam | Nov 2000 | A |
| 6153581 | Sanaka | Nov 2000 | A |
| 6162907 | Habener | Dec 2000 | A |
| 6165470 | Becquart et al. | Dec 2000 | A |
| 6171828 | Magota et al. | Jan 2001 | B1 |
| 6172046 | Albrecht et al. | Jan 2001 | B1 |
| 6191102 | DiMarchi et al. | Feb 2001 | B1 |
| 6193997 | Modi | Feb 2001 | B1 |
| 6201072 | Rathi et al. | Mar 2001 | B1 |
| 6214547 | Kjeldsen et al. | Apr 2001 | B1 |
| 6214863 | Bissery | Apr 2001 | B1 |
| 6217893 | Pellet et al. | Apr 2001 | B1 |
| 6221378 | Modi | Apr 2001 | B1 |
| 6221958 | Shalaby et al. | Apr 2001 | B1 |
| 6231882 | Modi | May 2001 | B1 |
| 6239167 | Bissery | May 2001 | B1 |
| 6242479 | Wechter | Jun 2001 | B1 |
| RE37302 | Efendic et al. | Jul 2001 | E |
| 6258377 | New et al. | Jul 2001 | B1 |
| 6271200 | Modi | Aug 2001 | B1 |
| 6277819 | Efendic | Aug 2001 | B1 |
| 6284725 | Coolidge et al. | Sep 2001 | B1 |
| 6284727 | Kim et al. | Sep 2001 | B1 |
| 6287588 | Shih et al. | Sep 2001 | B1 |
| 6290987 | Modi | Sep 2001 | B1 |
| 6294153 | Modi | Sep 2001 | B1 |
| 6299872 | Albrecht et al. | Oct 2001 | B1 |
| 6312665 | Modi | Nov 2001 | B1 |
| 6316224 | Xia | Nov 2001 | B1 |
| 6329336 | Bridon et al. | Dec 2001 | B1 |
| 6340742 | Burg et al. | Jan 2002 | B1 |
| 6346543 | Bissery et al. | Feb 2002 | B1 |
| 6348192 | Chan et al. | Feb 2002 | B1 |
| 6348327 | Gorman et al. | Feb 2002 | B1 |
| 6387365 | Albrecht et al. | May 2002 | B1 |
| 6448225 | O'Connor et al. | Sep 2002 | B1 |
| 6461605 | Cutler et al. | Oct 2002 | B1 |
| 6472373 | Albrecht | Oct 2002 | B1 |
| 6482613 | Goeddel et al. | Nov 2002 | B1 |
| 6514500 | Bridon et al. | Feb 2003 | B1 |
| 6569832 | Knudsen et al. | May 2003 | B1 |
| 6583111 | DiMarchi et al. | Jun 2003 | B1 |
| 6610830 | Goeddel et al. | Aug 2003 | B1 |
| 20010002394 | Efendic et al. | May 2001 | A1 |
| 20010006943 | Jensen et al. | Jul 2001 | A1 |
| 20010011071 | Knudsen et al. | Aug 2001 | A1 |
| 20010014666 | Hermeling et al. | Aug 2001 | A1 |
| 20010021767 | Drucker et al. | Sep 2001 | A1 |
| 20010046956 | Hadock | Nov 2001 | A1 |
| 20020037841 | Papadimitriou | Mar 2002 | A1 |
| 20020048571 | Gyuris et al. | Apr 2002 | A1 |
| 20020106719 | Choi et al. | Aug 2002 | A1 |
| 20020193570 | Gillies et al. | Dec 2002 | A1 |
| 20030108567 | Bridon et al. | Jun 2003 | A1 |
| 20030108568 | Bridon et al. | Jun 2003 | A1 |
| 20040063635 | Yu et al. | Apr 2004 | A1 |
| 20040121426 | Hsieh | Jun 2004 | A1 |
| Number | Date | Country |
|---|---|---|
| 704594 | May 1995 | AU |
| 741964 | Nov 1998 | AU |
| 2022539 | Feb 1991 | CA |
| 2270320 | Oct 1999 | CA |
| 2309810 | May 2000 | CA |
| 1341211 | Mar 2001 | CA |
| 1235981 | Nov 1999 | CN |
| 1239103 | Dec 1999 | CN |
| 37 23 781 | Jan 1988 | DE |
| 19921537 | Nov 2000 | DE |
| 0 028 033 | May 1981 | EP |
| 0 032 134 | Jul 1981 | EP |
| 0 048 970 | Apr 1982 | EP |
| 0 068 701 | Jan 1983 | EP |
| 0 070 906 | Feb 1983 | EP |
| 0 073 646 | Mar 1983 | EP |
| 0 079 739 | May 1983 | EP |
| 0 088 632 | Sep 1983 | EP |
| 0 091 527 | Oct 1983 | EP |
| 0 116 201 | Aug 1984 | EP |
| 0 123 294 | Oct 1984 | EP |
| 0 123 544 | Oct 1984 | EP |
| 0 138 437 | Apr 1985 | EP |
| 0 146 413 | Jun 1985 | EP |
| 0 147 198 | Jul 1985 | EP |
| 0 163 406 | Dec 1985 | EP |
| 0 172 619 | Feb 1986 | EP |
| 0 106 179 | May 1986 | EP |
| 0 196 056 | Oct 1986 | EP |
| 0 201 239 | Nov 1986 | EP |
| 0 206 733 | Dec 1986 | EP |
| 0 215 658 | Mar 1987 | EP |
| 0 236 210 | Sep 1987 | EP |
| 0 237 019 | Sep 1987 | EP |
| 0 241 435 | Oct 1987 | EP |
| 0 244 221 | Nov 1987 | EP |
| 0 252 561 | Jan 1988 | EP |
| 0 169 566 | Jun 1988 | EP |
| 0 301 670 | Feb 1989 | EP |
| 0 308 381 | Mar 1989 | EP |
| 0 314 317 | May 1989 | EP |
| 0 077 670 | Jun 1989 | EP |
| 0 319 641 | Jun 1989 | EP |
| 0 322 094 | Jun 1989 | EP |
| 0 325 262 | Jul 1989 | EP |
| 0 330 451 | Aug 1989 | EP |
| 0 218 825 | Sep 1989 | EP |
| 0 339 568 | Nov 1989 | EP |
| 0 344 459 | Dec 1989 | EP |
| 0 361 991 | Apr 1990 | EP |
| 0 366 400 | May 1990 | EP |
| 0 041 313 | Sep 1990 | EP |
| 0 395 918 | Nov 1990 | EP |
| 0 399 666 | Nov 1990 | EP |
| 0 163 406 | Jan 1991 | EP |
| 0 413 622 | Feb 1991 | EP |
| 0 416 673 | Mar 1991 | EP |
| 0 205 564 | May 1991 | EP |
| 0 237 545 | May 1991 | EP |
| 0 230 980 | Jul 1991 | EP |
| 0 230 980 | Jul 1991 | EP |
| 0 118 617 | Aug 1991 | EP |
| 0 163 529 | Aug 1991 | EP |
| 0 267 208 | Aug 1991 | EP |
| 0 195 691 | Nov 1991 | EP |
| 0 121 884 | Jan 1992 | EP |
| 0 217 404 | Jan 1992 | EP |
| 0 231 819 | Apr 1992 | EP |
| 0 209 539 | May 1992 | EP |
| 0 503 583 | Sep 1992 | EP |
| 0 509 841 | Oct 1992 | EP |
| 0 510 678 | Oct 1992 | EP |
| 0 510 693 | Oct 1992 | EP |
| 0 022 242 | Nov 1992 | EP |
| 0 511 912 | Nov 1992 | EP |
| 0 229 016 | Dec 1992 | EP |
| 0 241 435 | Dec 1992 | EP |
| 0 364 980 | Apr 1993 | EP |
| 0 317 254 | Sep 1993 | EP |
| 0 347 781 | Feb 1994 | EP |
| 0 422 697 | Mar 1994 | EP |
| 0 619 322 | Oct 1994 | EP |
| 0 658 568 | Jun 1995 | EP |
| 0 300 466 | Sep 1995 | EP |
| 0 222 279 | Jan 1996 | EP |
| 0 146 354 | Mar 1996 | EP |
| 0 401 384 | Mar 1996 | EP |
| 0 711 835 | May 1996 | EP |
| 0 427 296 | Sep 1996 | EP |
| 0 741 188 | Nov 1996 | EP |
| 0 751 220 | Jan 1997 | EP |
| 0 640 619 | Jul 1997 | EP |
| 0 771 871 | Jul 1997 | EP |
| 0 201 239 | Oct 1998 | EP |
| 0 734 450 | Jan 1999 | EP |
| 0 741 188 | Jul 1999 | EP |
| 0 736 303 | Aug 1999 | EP |
| 0 764 209 | Jan 2001 | EP |
| 1 099 441 | May 2001 | EP |
| 1 125 579 | Aug 2001 | EP |
| 0 903 148 | Oct 2001 | EP |
| 0 956 861 | Apr 2002 | EP |
| 1 213 029 | Jun 2002 | EP |
| 1 136 075 | Jan 2003 | EP |
| 0 946 191 | Mar 2003 | EP |
| 0 889 949 | May 2003 | EP |
| 1 317 929 | Jun 2003 | EP |
| 2 635 115 | Sep 1990 | FR |
| 2 719 593 | Nov 1995 | FR |
| 2 193 631 | Feb 1988 | GB |
| 2 350 362 | Nov 2000 | GB |
| 1 117790 | May 1989 | JP |
| 2 117384 | May 1990 | JP |
| 2 227079 | Sep 1990 | JP |
| 3 27320 | Feb 1991 | JP |
| 3 201987 | Sep 1991 | JP |
| 4 211375 | Aug 1992 | JP |
| 5 292972 | Nov 1993 | JP |
| 6-22784 | Feb 1994 | JP |
| 6 38771 | Feb 1994 | JP |
| 8-51982 | Feb 1996 | JP |
| 8 53500 | Feb 1996 | JP |
| 8 59509 | Mar 1996 | JP |
| WO 8202715 | Aug 1982 | WO |
| WO 8302461 | Jul 1983 | WO |
| WO 8503079 | Jul 1985 | WO |
| WO 8703887 | Jul 1987 | WO |
| WO 8902922 | Apr 1989 | WO |
| WO 9001063 | Feb 1990 | WO |
| WO 9001540 | Feb 1990 | WO |
| WO 9004788 | May 1990 | WO |
| WO 9011296 | Oct 1990 | WO |
| WO 9013653 | Nov 1990 | WO |
| WO 9102754 | Mar 1991 | WO |
| WO 9105052 | Apr 1991 | WO |
| WO 9108220 | Jun 1991 | WO |
| WO 9111457 | Aug 1991 | WO |
| WO 9201055 | Jan 1992 | WO |
| WO 9300109 | Jan 1993 | WO |
| WO 9300109 | Jan 1993 | WO |
| WO 9300437 | Jan 1993 | WO |
| WO 9303164 | Feb 1993 | WO |
| WO 9315199 | Aug 1993 | WO |
| WO 9315200 | Aug 1993 | WO |
| WO 9315211 | Aug 1993 | WO |
| WO 9318785 | Sep 1993 | WO |
| WO 9318786 | Sep 1993 | WO |
| WO 9325579 | Dec 1993 | WO |
| WO 9419373 | Sep 1994 | WO |
| WO 9424160 | Oct 1994 | WO |
| WO 9425489 | Nov 1994 | WO |
| WO 9503405 | Feb 1995 | WO |
| WO 9505465 | Feb 1995 | WO |
| WO 9505848 | Mar 1995 | WO |
| WO 9512684 | May 1995 | WO |
| WO 9516708 | Jun 1995 | WO |
| WO 9517510 | Jun 1995 | WO |
| WO 9523857 | Sep 1995 | WO |
| WO 9527059 | Oct 1995 | WO |
| WO 9530759 | Nov 1995 | WO |
| WO 9531214 | Nov 1995 | WO |
| WO 9603144 | Feb 1996 | WO |
| WO 9608512 | Mar 1996 | WO |
| WO 9614409 | May 1996 | WO |
| WO 9614416 | May 1996 | WO |
| WO 9617941 | Jun 1996 | WO |
| WO 9617942 | Jun 1996 | WO |
| WO 9618412 | Jun 1996 | WO |
| WO 9620005 | Jul 1996 | WO |
| WO 9707814 | Mar 1997 | WO |
| WO 9715296 | May 1997 | WO |
| WO 9724445 | Jul 1997 | WO |
| WO 9726321 | Jul 1997 | WO |
| WO 9731943 | Sep 1997 | WO |
| WO 9734997 | Sep 1997 | WO |
| WO 9739132 | Oct 1997 | WO |
| WO 9749729 | Dec 1997 | WO |
| WO 9800158 | Jan 1998 | WO |
| WO 9804718 | Feb 1998 | WO |
| WO 9808531 | Mar 1998 | WO |
| WO 9808873 | Mar 1998 | WO |
| WO 9811136 | Mar 1998 | WO |
| WO 9819698 | May 1998 | WO |
| WO 9820895 | May 1998 | WO |
| WO 9847489 | Jul 1998 | WO |
| WO 9836085 | Aug 1998 | WO |
| WO 9832867 | Oct 1998 | WO |
| WO 9900504 | Jan 1999 | WO |
| WO 9911781 | Mar 1999 | WO |
| WO 9913914 | Mar 1999 | WO |
| WO 9915193 | Apr 1999 | WO |
| WO 9915194 | Apr 1999 | WO |
| WO 9928346 | Jun 1999 | WO |
| WO 9929336 | Jun 1999 | WO |
| WO 9930731 | Jun 1999 | WO |
| WO 9940788 | Aug 1999 | WO |
| WO 9943706 | Sep 1999 | WO |
| WO 9947160 | Sep 1999 | WO |
| WO 9947161 | Sep 1999 | WO |
| WO 9953064 | Oct 1999 | WO |
| WO 9959621 | Nov 1999 | WO |
| WO 9964060 | Dec 1999 | WO |
| WO 9964061 | Dec 1999 | WO |
| WO 9966054 | Dec 1999 | WO |
| WO 0001727 | Jan 2000 | WO |
| WO 0004171 | Jan 2000 | WO |
| WO 0007617 | Feb 2000 | WO |
| WO 0009666 | Feb 2000 | WO |
| WO 0012116 | Mar 2000 | WO |
| WO 0016797 | Mar 2000 | WO |
| WO 0023459 | Apr 2000 | WO |
| WO 0024893 | May 2000 | WO |
| WO 0026354 | May 2000 | WO |
| WO 0032772 | Jun 2000 | WO |
| WO 0037051 | Jun 2000 | WO |
| WO 0037098 | Jun 2000 | WO |
| WO 0044772 | Aug 2000 | WO |
| WO 0062759 | Oct 2000 | WO |
| WO 0066138 | Nov 2000 | WO |
| WO 0066142 | Nov 2000 | WO |
| WO 0069911 | Nov 2000 | WO |
| WO 0069913 | Nov 2000 | WO |
| WO 0077039 | Dec 2000 | WO |
| WO 0078333 | Dec 2000 | WO |
| WO 0102017 | Jan 2001 | WO |
| WO 0105826 | Jan 2001 | WO |
| WO 0121602 | Mar 2001 | WO |
| WO 0127128 | Apr 2001 | WO |
| WO 0129242 | Apr 2001 | WO |
| WO 0130320 | May 2001 | WO |
| WO 0132200 | May 2001 | WO |
| WO 0136489 | May 2001 | WO |
| WO 0139784 | Jun 2001 | WO |
| WO 0151093 | Jul 2001 | WO |
| WO 0155213 | Aug 2001 | WO |
| WO 0157084 | Aug 2001 | WO |
| WO 0168112 | Sep 2001 | WO |
| WO 0181405 | Nov 2001 | WO |
| WO 0187322 | Nov 2001 | WO |
| WO 0198331 | Dec 2001 | WO |
| WO 0222151 | Mar 2002 | WO |
| WO 0245712 | Jun 2002 | WO |
| WO 0246227 | Jun 2002 | WO |
| WO 0247716 | Jun 2002 | WO |
| WO 0248192 | Jun 2002 | WO |
| WO 02066062 | Aug 2002 | WO |
| WO 02069994 | Sep 2002 | WO |
| WO 0270549 | Sep 2002 | WO |
| WO 02080676 | Oct 2002 | WO |
| WO 02085406 | Oct 2002 | WO |
| WO 02098348 | Dec 2002 | WO |
| WO 03002136 | Jan 2003 | WO |
| WO 03003971 | Jan 2003 | WO |
| WO 03011892 | Feb 2003 | WO |
| WO 03013573 | Feb 2003 | WO |
| WO 03014318 | Feb 2003 | WO |
| WO 03018516 | Mar 2003 | WO |
| WO 03076567 | Sep 2003 | WO |
| WO05000892 | Jan 2005 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20030082747 A1 | May 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09984186 | Oct 2001 | US |
| Child | 10237624 | US | |
| Parent | 08797689 | Jan 1997 | US |
| Child | 09258532 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09258532 | Feb 1999 | US |
| Child | 09984186 | US | |
| Parent | 08256927 | Jul 1994 | US |
| Child | 08797689 | US |
